Many positive-sense, single-stranded, RNA viruses infecting either plants or animals use ribosome recoding and other forms of non-canonical translation to produce important viral proteins such as the RNA dependent RNA polymerase (RdRp). In addition to viruses, evidence is emerging that ribosome recoding may be more widespread in eukaryotic genomes than initially thought and could have important implications for human diseases. Recoding events occur at a specific frequency and are critical for maintaining efficient replication. Previous animal virus recoding research has focused only on a small region of RNA surrounding the recoding site, assuming that all critical RNA structures are located in that small stretch of RNA. However, work using model plant viruses, including Turnip crinkle virus (TCV), has shown that RNA elements far outside of the recoding region play a critical role in recoding efficiency. Here, the importance of alternative RNA structures and long-range RNA:RNA interactions involving recoding sequences will be determined for Encephalomyocarditis virus (EMCV). EMCV is in the cardiovirus genus of Picornaviridae which includes the recently discovered human pathogen, Saffold virus. TCV will also be used to find novel internal ribosome entry sites, or IRES, which likely promote expression of the coat protein and novel isoforms of the RdRp. These studies will increase our understanding of ribosome recoding and IRES function in small RNA viruses. I will address the following questions in the proposed studies: 1) Are long-range RNA:RNA interactions required for efficient recoding in EMCV? Using full-length genome constructs, an in vitro and in vivo translation reporter assay will be developed to determine the effects of disrupting predicted long-range interactions on frameshifting. Reverse genetics will be used to determine if disrupting long-range RNA:RNA interactions is detrimental for virus accumulation. 2) Do alternative RNA structures in the EMCV recoding region exist? Critical alternative recoding structures exist for TCV and similar structures are predicted by folding algorithms to exist for EMCV. SHAPE RNA structure probing will be used to determine the structure of the EMCV recoding region both in vitro and in vivo. The importance of alternative structures in recoding will be determined using both in vitro and in vivo translation assays. 3) Locate novel IRES in TCV. The coat protein and novel RdRp isoforms are expressed from internal initiation in in vitro translation assays. The IRES sequences responsible for either coat protein or RdRp isoform expression will be determined experimentally using SHAPE, mutagenesis, and in vitro translation assays. The importance of RdRp isoforms for TCV accumulation will also be determined. The proposed studies will greatly benefit the ribosome recoding and IRES-related fields by taking an innovative approach that will bridge animal and plant virus studies.